Work in this laboratory has already established that wall lytic enzymes are required for cell growth in the bacterium Bacillus subtilis. We propose to isolate temperature sensitive mutations in each of the two lytic enzymes of this bacterium to elucidate the exact effect of lytic enzyme deprivation. The mutants will be characterized using light microscopic, electron microscopic and biochemical techniques. We have recently developed a Bacillus megaterium bacterial wall synthetic system using membrane vesicles which makes crosslinked peptidoglycan of size large enough to be visible electron microscopically. We propose to carry out the visualization of such in vitro made all walls to study their spatial orientation. Experiments in the literature indicate that mutants can be found in which membrane bound enzymes are released into solution. We propose to isolate such mutants in wall synthetic and other membrane associated enzymes. We hope to be able to add such enzymes to other mutants lacking these enzymes in order to phenotypically reverse the deficiency.